The field of the present invention is that of food supplements which are commonly prepared from soybeans or soybean meal. The factors of the nutritive qualities of such foodstuffs and their cost of preparation are significant in determining their commercial success and therefore commercial availability. Specifically, the invention concerns methods for improving the quality and the cost efficiency of preparation of foodstuff supplements by overcoming the problem of trypsin inhibition due to a protein native to the soybean preparation.
Trypsin is an important digestive enzyme, particularly in certain species where ancillary enzymes, such as pepsin and chymotrypsin are present in relatively small amounts, or are absent. From an economic standpoint, the most important of these species are chickens, pigs, and calves (when the calves are sufficiently young that they have not developed a fully mature digestive system). In such animals, in particular, if the enzyme trypsin is in some way impaired in its functioning, there are a number of deleterious results. First, any food which is ingested by the animal is lowered in nutritive value because of a directly impaired capacity to digest it. Second, even in animals which contain other digestive enzymes in addition to trypsin, trypsin normally activates some of these enzymes and allows their participation in the process. A deficiency in trypsin thus results in a concomitant deficiency in these enzymes. Finally, in response to a perceived lack of adequate trypsin, the pancreas is induced to release more trypsin than it is capable easily of releasing, resulting in an "overwork" condition called pancreatic hypertrophy, which, at best, results in morbidity and, at worst, in death.
Trypsin inhibition is an insurmountable problem when the ingested foodstuff contains large quantities of soybean materials which have not been subjected to proper treatment to destroy a soybean trypsin inhibitor (STI) which inhibitor is capable of binding the endogenous trypsin in the animal ingesting the foodstuff, and in preventing it from carrying out its normal function. Hence, animal foods which are largely soybean based are currently treated by "cooking" to inactivate this protein. In conventional soy processing, the soybeans are dehulled using a wet process, wherein the water content, however, is purposely limited in order to reduce waste weight and in order to prevent interference with subsequent processing steps. The hulled soybeans are then extracted with hexane to remove the soybean oil for commercial use. After the hexane extraction, the soybean mulch is heated to inactivate the soybean trypsin inhibitor.
This inactivation process is conducted at considerable expense, and with imperfect results. As would be expected, the heating produces a decline in STI content which exponentially decays to a curve assymptotically approaching zero. Therefore, after a time period which is optimum for the particular preparation in question, further heating becomes uneconomic and counterproductive even though additional amounts of STI would be thereby inactivated. The resulting processed soybean meal is then used in animal feeds in a variety of forms, and is reduced in STI but still contains residual amounts.
The most common use of this preparation is as a feed additive which is added to other carbohydrate sources used for livestock feeding, the most important livestock types being pigs and chickens, as well as newborn calves. However, as fed to calves, the preparation is more commonly used as a milk replacement by suspending the preparation in a liquid before feeding. This is formulated either as a solid which may subsequently be made up in liquid form by the livestock raiser, or as a liquid concentrate which is diluted before feeding. Although the constituency is smaller in number, soybean preparations are also used as feeding supplements for human infants, particularly those who exhibit an intolerance for milk products.
The problem of trypsin inhibition has also been studied from a purely research viewpoint. It is known that STI reacts with bovine trypsin by specifically binding to the reactive site of the trypsin; the STI itself being hydrolyzed at the interface due to the action of the inhibited trypsin itself. (Laskowski and Sealock, Enzymes, 3rd edition, 375 (1971); Finkenstadt et al., Proceedings of the International Conference of Proteinase Inhibitors, Second, 389 (1974). The mechanism of this inhibition is reasonably well understood. Mattis and Laskowski, Biochemistry 12: 2239 (1973); Ruhlmann et al., Journal of Molecular Biology, 77: 417 (1973); Huber et al., Journal of Molecular Biology, 89: 73 (1974), and Sweet et al., Biochemistry 13: 4212 (1974). It appears that a fairly tight complex is formed between the inhibitor and the trypsin.
Incubation of catalytic amounts of trypsin with STI results in specific hydrolysis of a single peptide bond, the reactive site. This hydrolysis leads to the establishment of an equilibrium between virgin (reactive site peptide bond intact) and modified (reactive site peptide bond hydrolyzed) inhibitor. Both virgin and modified STI are inhibitors of trypsin, so this hydrolysis will not by itself inactivate STI. Once the modified inhibitor is formed, however, it can be inactivated by further reaction with carboxypeptidase B. This enzyme removes an amino acid (arginine 163) from modified STI. The product of this reaction (des-Arg STI) is no longer capable of inhibiting trypsin. Although trypsin can catalyze the conversion of virgin to modified STI, it does so at such a slow rate (several days at neutral pH) that trypsin cannot be used effectively to inactivate STI.
It has recently been shown, using purified STI, that a starfish, Dermasterias imbricata, contains a trypsin enzyme (DIT.sub.1) which is capable of converting virgin to modified STI at an extremely rapid rate (several minutes at neutral pH (Estell and Laskowski, Biochemistry 19: 124, 1980)). Thus, STI activity can be permanently destroyed by use of this enzyme in combination with another supplementary proteolytic enzyme, for example, carboxypeptidase B, which inactivates the modified STI.
The present invention, therefore, in providing DIT.sub.1, offers a solution to the problem of detraction due to the presence of soybean trypsin inhibitor (STI) from desirability of use of soybean as a food.